This invention relates to novel nonmammalian carrier vectors and viruses useful in the production of high titers of recombinant viruses which may contain foreign DNA inserts or which may be point-mutated or deleted viruses, and methods of producing those viruses. The nonmammalian carrier vector (xe2x80x9ccarrier vectorxe2x80x9d) is a chimeric vector which includes those portions of a nonmammalian virus backbone which allow replication in a nonmammalian host cell. The carrier vector includes various nucleic acid cassettes, which may include an embedded recombinant viral genome containing a desired transgene, components necessary for production of a replication-defective recombinant virus containing the transgene, and domains that permit the carrier vector to bind to mammalian cells. The invention also provides methods of producing high concentrations of recombinant virus as a substantially homogeneous preparation, compositions to produce the recombinant virus, and novel recombinant viruses.
A recombinant virus carrying a foreign DNA insert may be used to deliver genes to cells, where the gene may be expressed, if desired, to permit production of recombinant proteins in vitro or in vivo, vaccination of human and non-human mammals, or treatment or amelioration of diseases or genetic defects in humans or non-human mammals. One may treat or ameliorate diseases or genetic defects by providing normal gene products, increased levels of gene products or by blocking endogenous production of a gene, whose expression would be deleterious to the cell or organism.
Methods for delivering an exogenous gene to a mammalian cell include the use of mammalian viral vectors, such as those which are derived from retroviruses, adenoviruses, herpes viruses, vaccinia viruses, polio viruses, adeno-associated viruses, hybrid viruses (e.g., hybrid adenovirus-AAV, see U.S. Pat. No. 5,856,152) and the like. Other methods include direct injection of DNA, biolistic administration of DNA, electroporation, calcium phosphate precipitation, as well as methods of administration which utilize ligand-DNA conjugates, liposome conjugates of DNA, polycation-DNA complexes or adenovirus-ligand-DNA conjugates.
A transgene is a nucleic acid encoding a protein of interest; it may be a gene to allow for genetic or drug selection, e.g., a gene conferring resistance to antibiotics, or a reporter gene allowing detection, e.g., by color in the case of the use of green fluorescent protein. Alternatively, the transgene may be one that is useful for corrective applications. For instance, a transgene may be a normal gene that replaces or augments the function of a patient""s defective gene. The transgene may be one that counteracts the effects of a disease, such as introduction and expression of a gene that is distinct from the one that it replaces or augments, but which has the same function or compensates for the defective gene""s function. The transgene may be a gene which blocks or represses the expression of a malfunctioning, mutated, or viral gene in the patient, thereby giving rise to a corrective effect. A transgene may also be used for immunization against various agents, by provoking an immunogenic response in an animal. Delivery of therapeutic transgenes to a patient thus effects a correction of a defect or prevention of disease. The transgene also may be one which is useful for production of proteins in vitro, such as for large-scale production of therapeutic proteins.
Appropriate genes for expression in the cell include, without limitation, those genes which are normally expressed in cells but whose products are produced in insufficient amounts. Alternatively, the appropriate gene for expression is one which expresses a normal gene product which replaces a defective gene product, encodes ribozymes or antisense molecules which repair or destroy mutant cellular RNAs expressed from mutated genes, or modifies or destroys viral RNAs. Transgenes used for production of proteins in vitro include proteins such as secreted factors, including hormones, growth factors and enzymes.
Many gene therapy methods involve supplying an exogenous gene to overcome a deficiency in the expression of a gene in a patient. Some of these deficiencies are congenital and are due to a mutation in a particular gene in all the cells of the patient. For instance, in cystic fibrosis, there are one or more mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) which prevents the CFTR protein from functioning properly. In other cases, a deficiency in gene expression is due to an accident or disease that occurs during the patient""s life. For instance, in Type I diabetes mellitus, the xcex2 pancreatic islet cells, which produce insulin, are destroyed, such that patients with this disease can no longer synthesize insulin. In other cases, the endogenous gene may be structurally normal but is not produced in high enough quantities due to disease, medical treatment or other environmental conditions, or mutations in the regulatory elements of the endogenous gene. For example, there are a number of blood disorders, such as anemia, in which there is insufficient production of red blood cells, which may be treated with erythropoietin (EPO) or with a transgene encoding EPO. Transgenes may also be used for genetic immunization, i.e., to elicit an immune response to a pathogen in an animal, including humans. For instance, a transgene may include a sequence from a viral, bacterial or fungal pathogen, such as influenza virus, human immunodeficiency virus (HIV), or mycobacterium tuberculosis.
Certain methods are amenable to targeted delivery of the exogenous gene to specific tissues, such as liver tissue. One method of delivering genes to specific cells relies upon the function of a cell-specific receptor. The asialoglycoprotein receptor (ASGP-R), which is present on the surface of hepatocytes (Spiess et al., 1990, Biochem. 29:10009-10018), is a lectin which has affinity for the terminal galactose residues of glycoproteins, and has been used to target gene delivery to liver hepatocytes. For example, a DNA complex is bound to a ASGP-R on the cell surface, allowing subsequent endoyctosis by the liver hepatocyte.
Viruses that are commonly used in gene delivery applications are modified by replacing viral nucleic acid with a desired transgene. Frequently, DNA removed from the virus encodes proteins necessary for viral replication or encapsidation, in which case the recombinant virus containing a transgene is replication-deficient and will not replicate or encapsidate in the host. To permit replication and encapsidation, current methods recognize that those portions of DNA which have been deleted must be supplied by wild-type or modified viruses or by plasmids containing DNA encoding the required gene products. Supplying wild-type or modified virus may result in recombinant virus stocks contaminated with wild-type or modified virus. Supplying plasmids encoding the required gene products through cotransfection results in low efficiency of recombinant virus production, as well as recombination events which yield wild-type virus contaminants.
A number of different viruses have been used to deliver a transgene to mammalian cells. These viruses include retrovirus, hepatitis B virus (HBV), adenovirus, adeno-associated virus (AAV) and herpesvirus. AAV possesses unique features that make it attractive as a vector for delivering foreign DNA (i.e., a transgene) to cells, and various groups have studied the potential use of AAV in the treatment of disease states.
AAV is a parvovirus, the genome of which is about 4.7 kb in length, including 145 nucleotide inverted terminal repeats (ITRs). The AAV genome encodes two genes, rep and cap, each of which expresses a family of related proteins from separate open reading frames and produced as a result of alternative mRNA splicing. Rep polypeptides (rep78, rep68, rep52, and rep40) are involved in replication, rescue and integration of the AAV genome. Cap proteins (VP1, VP2, and VP3) form the virion capsid. Flanking the rep and cap open reading frames at the 5xe2x80x2 and 3xe2x80x2 ends of the AAV genome are the 145 bp ITRs, the first 125 bp of which are capable of forming Y- or T-shaped duplex structures. The entire nucleic acid encoding rep and cap can be excised and replaced with a transgene [B. J. Carter, in xe2x80x9cHandbook of Parvovirusesxe2x80x9d, ed., P. Tijsser, CRC Press, pp. 155-168 (1990)]. The ITRs represent the minimal sequence required for replication, rescue, packaging, and integration of the AAV genome.
When this nonpathogenic human virus infects a human cell, the viral genome integrates into chromosome 19 resulting in latent infection of the cell. Production of infectious virus and replication of the virus does not occur unless the cell is coinfected with a lytic helper virus, such as adenovirus (Ad) or herpesvirus. Upon infection with a helper virus, the AAV provirus is rescued and amplified, and both AAV and helper virus are produced. The infecting parental ssDNA is converted to duplex replicating form (RF) DNAs in a rep dependent manner. The rescued AAV genomes are packaged into preformed protein capsids (icosahedral symmetry approximately 20 nm in diameter) and released as infectious virions that have packaged either+orxe2x88x92ss DNA genomes following cell lysis. However, progress towards establishing AAV as a transducing vector for the delivery of DNA in the form of a desired transgene has been slow for a variety of reasons.
Replacing the rep and cap sequences with a desired transgene yields a recombinant virus capable of delivering the transgene to target host cells. However, because AAV requires a particular genome packaging size, addition of a transgene results in deletion of necessary gene functions for rep and cap. In current methods, necessary gene functions replaced by the transgene are supplied by viruses or additional plasmids. Furthermore, the requirement by AAV for helper virus functions also requires the use of helper viruses (either wildtype or crippled viruses) or plasmids containing the helper virus functions.
One method that has been used to produce recombinant AAV (rAAV) vectors comprises co-transfecting eukaryotic cells with a plasmid containing rAAV (the cis plasmid) and a plasmid containing rep and cap (the trans plasmid), and infecting the cells with a helper virus (e.g., adenovirus or herpes virus). See U.S. Pat. No. 5,753,500. The disadvantage of this method is that the rAAV vector stock is contaminated with helper virus, which is labor-intensive and difficult to separate from the helper virus, and co-transfection of two plasmids along with infection by a helper virus is inefficient and cannot be easily scaled up for industrial production of rAAV.
A second method that has been used to produced rAAV involves a triple plasmid transfection of eukaryotic cells. In this method, one plasmid carries the transgene and ITRs (the cis plasmid), a second plasmid encodes the rep and cap genes (the trans plasmid), and the third plasmid encodes the helper virus functions, i.e. adenoviral genes such as E1a, E1b, E2a and E4 (the helper plasmid). The disadvantage of this method is that a triple transfection is also inefficient and is difficult to scale up.
A third method involves the use of a packaging cell line such as one including AAV functions rep and cap. See U.S. Pat. No. 5,658,785 and U.S. Ser. No. PCT US98/19463. The packaging cell line may be transfected with a cis plasmid comprising the transgene and ITRs, and infected by wild-type adenovirus (Ad) helper. See U.S. Pat. No. 5,658,785. Alternatively, the packaging cell line may be co-infected by a hybrid Ad/AAV, in which a hybrid Ad vector caries the cis plasmid in the E1 locus (see U.S. Pat. No. 5,856,152), and by a wild-type or mutant Ad that supplies E1. The disadvantage of this method is that wild-type Ad may be produced, which must be separated from the rAAV vector before use in a patient.
Thus, current methods of producing recombinant AAV are incapable of yielding the high amounts of essentially homogeneous virus for pharmaceutical compositions needed for the treatment of a large number of patients in a easily scaled industrial production.
Nonmammalian viruses have been used to transiently express particular individual exogenous proteins in either mammalian or non-mammalian cells. For example, viruses of the family Baculoviridae, or xe2x80x9cbaculovirusesxe2x80x9d, which normally infect members of the order Lepidoptera, have been used to express exogenous genes in insect cells. Baculoviruses have also been reported to enter mammalian cells, and baculoviral DNA has been detected in nuclear extracts of mammalian cells (Volkman et al., 1983, Appl. Environ. Microbiol. 45:1085-1093). While one report of baculovirus-mediated gene expression in mammalian cells has appeared, the authors later attributed the apparent reporter gene activity to the reporter gene product being carried into the cell after a prolonged incubation of the cell with the virus (Carbonell et al., 1987, Appl. Environ. Microbiol. 53:1412-1417). These authors reported that, when the exogenous gene gains access to the cell as part of the baculovirus genome, the exogenous gene is not expressed de novo. Subsequent studies have demonstrated baculovirus-mediated gene expression of particular proteins in mammalian cells (Boyce et al., 1996, Proc. Natl. Acad. Sci. USA, 93:2348-2352).
While baculovirus has been used for expressing particular proteins in a mammalian cell, see U.S. Pat. No. 5,731,182, baculovirus has not been used to produce pharmaceutical compositions of replication-deficient recombinant virus using an easily scaled industrial process. As disclosed in U.S. Pat. No. 5,731,182, the genome of the baculovirus may be modified by insertion of ligand DNA, which comprises a gene encoding a mammalian receptor specific protein that allows the baculovirus to bind and enter mammalian cells. The nonmammalian virus infecting the mammalian cells allows only for transient expression of the transgene within the mammalian cell. In addition, the methods disclosed in U.S. Pat. No. 5,731,182 do not result in production of an altogether distinct, essentially homogeneous recombinant virus, at high titers.
The problem of generating recombinant replication-deficient virus that is produced in the absence of helper viruses and by an efficient method that is applicable to large-scale industrial production has not been solved until the present invention. Current viral production methods include costly and time consuming purification and concentration steps, and are incapable of producing sufficient recombinant virus for pharmaceutic applications. In the case of AAV, for example, current methods produce at most on the order of 104-105 genomic copies (gc) of recombinant virus per producer cell. Similarly, current methods are suitable for producing recombinant adenovirus in amounts on the order of 104 particles per producer cell, and retroviruses in amounts on the order of 102-104 colony forming units (cfu) per producer cell. Current production methods result in contaminating helper virus which must be inactivated and/or removed from the final products prior to pharmaceutical application. Thus, there exists an unfulfilled need for a method of manufacturing recombinant mammalian virus at high titers free of other contaminating virus in order to produce recombinant viruses capable of delivering a desired transgene to mammalian cells, or immunizing cells against viral or bacterial infection by the use of such recombinant viruses, in a stable fashion.
The invention exploits the properties of nonmammalian and mammalian viruses to create novel chimeric vectors and viruses for the manufacture of an essentially homogeneous recombinant virus preparation in the absence of contaminating helper virus using a process that may be easily scaled for industrial production. The essentially homogeneous recombinant virus may be used for various purposes, including delivering a desired transgene to mammalian cells for pharmaceutic applications including immunization and correction of genetic defects; transient and stable gene transfer in vivo, in vitro and ex vivo; production of proteins in vivo or in vitro; and other methods in which high levels of gene transduction into a cell are required, e.g., in the production of expression libraries for screening compounds or for introducing genes into cells that are not easily transfected.
The carrier vector of the invention is a chimeric vector backbone derived from the nucleic acid of a nonmammalian virus, and includes one or more of the following elements: 1) an embedded recombinant viral genome; 2) nucleic acid sequences which encode proteins required for replication and encapsidation of the recombinant virus genome; 3) nucleic acid sequences encoding helper functions (if the recombinant virus to be produced is helper-dependent, e.g., AAV); 4) nucleic acid sequences encoding a ligand that can interact with a mammalian cell; and 5) regulatory control sequences that regulate nucleic acid sequences in the nonmammalian virus backbone or in a replication-deficient portion or modification thereof The carrier vector may also include any other nucleic acid sequences that are required to produce a replication-deficient recombinant virus.
In one embodiment of the invention, one or more carrier vectors may comprise all of the elements required to produce a replication-deficient recombinant vector in a particular host cell or cell line. The number and type of elements that are required will depend upon the particular host cell used and the type of recombinant vector produced. For instance, if a recombinant AAV vector is desired and the host cell line is one which has rep and cap stably integrated in its genome, the carrier vector or vectors would comprise 1) an embedded recombinant viral genome comprising the AAV ITRs and the transgene and 2) separate helper functions, which may include any nucleic acid sequence required for replication and encapsidation of the rAAV. For instance, these helper functions may include any one or a combination of E1, E2a, E4ORF6 and VAI from adenovirus (Ad). If a recombinant AAV vector is to be produced in a host cell line that does not express rep and cap, then the carrier vector or vectors may also include the DNA sequences encoding rep and cap.
Alternatively, if a recombinant retrovirus is desired, the carrier vector or vectors would comprise 1) an embedded recombinant viral genome comprising the retroviral LTRs and the transgene of interest driven from the retroviral LTRs or from a heterologous promoter, and 2) DNA sequences encoding any one or a combination of gag, pol and env for the functions of replication and encapsidation of the retrovirus not supplied in the host cell. In a preferred embodiment, all of the required elements to produce a recombinant virus in a particular host cell are contained on a single carrier vector because the use of a single carrier vector having all functions not supplied by a host cell increases the efficiency of transduction, and can be more easily scaled for industrial production of the embedded recombinant virus.
In an alternative embodiment, the carrier vector comprises an embedded recombinant viral genome, and any required replication, encapsidation and/or helper functions are provided by a helper virus or a plasmid.
The embedded recombinant viral genome may comprise a transgene and DNA elements required for replication of a mammalian virus. The transgene comprises the gene of interest, regulatory elements to regulate its expression, and an optional DNA spacer. The transgene is flanked by the DNA elements required for replication of a mammalian virus, such as the ITRs of AAV, the LTRs of retrovirus, or the ITRs of adenovirus. The recombinant viral genome is embedded within the nonmammalian virus backbone, optionally along with one or more of the other DNA sequences listed above, resulting in a chimeric carrier vector of the present invention.
In an alternative embodiment, the embedded recombinant viral genome does not contain a transgene but the recombinant viral genome itself contains point mutations or deletions. In this embodiment, the point mutations or deletions function to attenuate the replication of the subsequently-produced recombinant virus. The attenuated recombinant virus may be any virus which could be useful for vaccination, including, without limitation, picornaviruses such as poliovirus; hepatitis viruses such as hepatitis B and hepatitis C; cold-adapted respiratory syncytial virus (RSV); cold-adapted influenza virus; parainfluenza virus types 1, 2 and 3; and rotavirus.
The carrier vector is replication-proficient in its native host cells. For example, employing a baculovirus backbone results in a chimeric carrier vector that is replication-proficient in insect cells. In contrast, the embedded recombinant viral genome, optionally containing a transgene, is unable to excise, replicate, and package into virions because its promoters are inactive in insect cells. However, once the chimeric carrier vector infects a mammalian cell, the essential gene products required for replication and packaging of the carrier vector in its permissive native cell are no longer expressed. Thus, the carrier vector does not replicate in mammalian cells, and instead exists transiently within the mammalian cell.
In contrast, once the carrier vector has infected a mammalian cell, the mammalian regulatory sequences within the carrier vector controlling the embedded recombinant viral genome and other mammalian DNA sequences are activated, such that the recombinant viral genome is capable of being excised from the carrier vector and replicated. The capsid proteins which form the capsid of the recombinant virus are expressed such that the recombinant viral genome is encapsidated, which yields an infectious recombinant virus. The recombinant virus is essentially free of carrier vector because the carrier vector is not replicated in mammalian cells.
In a preferred embodiment, the recombinant virus is replication-deficient because there are no replication or helper functions present in the newly formed virions; i.e., the recombinant virus lacks part or all of the coding regions of the native virus genome. In embodiments of the invention in which the recombinant virus is helper-dependent, such as rAAV, the recombinant virus lacks both functional replication and encapsidation functions. In embodiments of the invention in which the recombinant virus is not helper-dependent, the recombinant virus lacks functional replication coding regions or other essential genes.
In cases where helper functions are required for recombinant virus production, recombinant virus may be produced without the need for coinfection and subsequent production of helper virus if a carrier vector includes the necessary helper functions. Thus, the invention yields lysates of substantially pure and essentially homogeneous preparations of the particular recombinant virus of interest in the absence of helper virus.
This invention thus has many advantages over current methods for manufacturing recombinant viruses. These advantages include: (1) the nonmammalian virus backbone permits insertion of large DNA sequences without compromising the efficiency of recombinant virus production; (2) sequences normally toxic to mammalian cells (e.g., AAV rep, VSV-G, retroviral envelope proteins, eukaryotic regulatory proteins, etc.) are not expressed in substantial amounts from their mammalian regulatory sequences in the nonmammalian host cell of the nonmammalian carrier vector and thus can be tolerated by the nonmammalian carrier vector during the course of its replication in the nonmammalian host cell; (3) nonmammalian viruses do not replicate in mammalian cells, precluding contamination of the final eukaryotic vector stocks with the nonmammalian carrier vector; (4) in some embodiments no helper viruses are necessary, with the result that the final recombinant virus preparation is essentially free of helper virus; (5) frequency of wildtype virus production due to homologous or non-homologous recombination is minimized; and (6) the methods of the present invention are particularly suitable to large scale production of recombinant viruses which are themselves replication-deficient. Additionally, nonmammalian viruses are not normally pathogenic to mammalian cells, may be propagated in serum free media, and may be grown to a high titer. Other features and advantages of the invention will be apparent from the following drawings, the description of the invention and its preferred embodiments, and the examples described herein.
In one embodiment, the present invention includes nonmammalian carrier vectors containing elements that are required to produce replication-deficient recombinant viral vectors. In a preferred embodiment, the nonmammalian carrier vector contains all the elements required to produce a replication-deficient recombinant viral vector. In an even more preferred embodiment, a single nonmammalian carrier vector contains all the required elements to produce a replication-deficient recombinant viral vector. In another preferred embodiment, the nonmammalian carrier vector is a baculovirus.
In another embodiment, the invention includes a method of producing replication-deficient recombinant viral vector lysates and stocks that are free of helper or other contaminating virus. In a preferred embodiment, the method is one which is easily scaled for industrial production of recombinant viral vectors. In another preferred embodiment, the method is one in which a high titer of recombinant viral vector lysates and stocks is achieved.
In another embodiment, the invention includes attenuated, replication-competent recombinant viruses and a method of producing such viruses free of helper or other contaminating virus. In a preferred embodiment, these attenuated, replication-competent viruses may be used for immunization.